Addition of Five Novel Fungal Flora to the Xylariomycetidae (Sordariomycetes, Ascomycota) in Northern Thailand

The deviation of conventional fungal niches is an important factor in the implications of hidden fungal diversity and global fungal numbers. The Xylariomycetidae (Sordariomycetes, Ascomycota), which is also referred to as xylarialean taxa, has a wide range of species that demonstrate a high degree of variation in their stromatic characteristics, showing either conspicuous or inconspicuous forms. In this study, samples were collected while focusing on temporal and spatial parameters and substrate characteristics. Based on internal transcribed spacer (ITS), 28S large subunit rDNA (LSU), RNA polymerase II second largest subunit (RPB2), and β-tubulin (TUB2) multigene phylogeny and morphology, five new species are introduced as Muscodor brunneascosporus, M. lamphunensis (Xylariaceae), Nigropunctata hydei, N. saccata (Incertae sedis), and Xenoanthostomella parvispora (Gyrotrichaceae). Plant substrates in the early stages of decay and attached to the host were feasible sample niches, with an emphasis on the collection of inconspicuous, hidden xylarialean species. The appearance of inconspicuous saprobic xylarialean forms during the rainy season may be linked to the change in nutritional mode, from endophytic mode during the dry season to saprobic in the wet. Therefore, it would be fascinating to concentrate future research on how seasonal fluctuations affect nutritional mode shifts, especially in northern Thailand, which would provide the optimal spatial characteristics. In order to establish a comprehensive linkage between endophytic and saprobic modes, it is imperative to have a substantial representation of endophytic isolate sequences resembling inconspicuous xylariaceous fungi within publicly accessible databases.


Introduction
A fungal stroma is a dense cluster of hyphal cells enveloped by a melanized outer layer of substances.Stromatic structures play a crucial role in the life cycle of various fungi, serving as the site for conidial and ascospore development [1].The characteristics of the substrate or the processes influencing growth are the primary drivers of stromal diversity [2].According to in vitro studies on Dussiella tuberiformis (Clavicipitaceae, Hypocreales), the development of the stromata is primarily influenced by non-reducing sugars, and the formation of the perithecia may be prompted by nutrition and moisture restriction [3].Stromata appear to have evolved convergently among ascomycetes and basidiomycetes, which act as a resilient propagule that can withstand harsh environmental conditions [1].Furthermore, stromata development is important for optimum temperature regulation for viable spores and maintaining the water content of the stromata to be able to spread spores, as well as for insects that need an appropriate temperature for their survival [4].Importantly, early fungal classifications have been focused on the presence or absence, nature, and interior development of like characters for the delimitation of systematic groups of major ranks [5].
The stromatic characters play a major role in the generic and family level classifications of species [6] in the Xylariomycetidae (referred to as "xylarialean taxa" herein).In certain xylarialean species, there is a specialized character known as the clypeus.It consists of stromatic tissue or melanized hyphae that form above the partially submerged or immersed ascomata, which are shield-shaped structures with variable development [2].Daranagama et al. [7] focused on those stromatic differences of xylarialean taxa and discussed the taxonomic significance of micro-xylariaceous (with inconspicuous ascomata) and macroxylariaceous (with conspicuous stromata) genera.Following seven stromatic characters among the xylarialean taxa, Samarakoon et al. [8] performed an ancestral character analysis.The results indicated that xylarialean taxa most likely diverged from inconspicuous forms when they first appeared.Additionally, the majority of the xylarialean taxa's undiscovered sexual morphs are presumably inconspicuous forms.However, there are limited studies on inconspicuous forms compared to conspicuous forms [7].
Taxonomic studies focusing on inconspicuous xylarialean taxa have been limited, probably due to a lack of fresh collections, and many species were described long ago and have never been recollected, especially from tropical locations [7].It is interesting to note that the genera transferred to new families were previously accepted but had uncertain morphologies and phylogenies.In contrast to conspicuous stromatic xylarialean taxa, those transferred taxa are morphologically unique in having inconspicuous, immersed ascomata that do not have key stromatic characters for delimiting higher ranks.Still, more than 50 incertae sedis genera, mostly inconspicuous forms, demanding fresh collections and taxonomic and phylogenetic studies [8].
Thailand is part of the Indo-Malayan hub of biodiversity, which is distinguished by a special combination of vascular plants and, as a result, many related species of fungi [10].Using polyphasic approaches and significant advancements in the hierarchical structure of classification, significant progress has been achieved in our understanding of the fungi in northern Thailand.Since 2017, our research has focused on the taxonomy, phylogeny, evolution, and secondary metabolites of xylarialean taxa in northern Thailand.In the provinces of Chiang Mai, Chiang Rai, Lampang, Nan, Phayao, and Phrae, we have introduced 20 new species and five new genera (Magnostiolata, Melanostictus, Neoamphisphaeria, Nigropunctata, and Paravamsapriya) until 2022 [8,[11][12][13].Based on our previous studies, there is a high possibility of the addition of new inconspicuous xylarialean species from northern Thailand.In this study, we introduce five new inconspicuous xylarialean species Fresh specimens were collected mainly during the rainy season in 2022 from northern Thailand.External examinations were carried out using a stereomicroscope (Leica M205 FCA-FA, Leica Microsystems Ltd., Hessian, Germany) and photographed with a Leica DMC6200 digital microscope camera.Microscopic photography was performed using a Nikon DS-Ri2 camera connected with a Nikon ECLIPSE Ni (Tokyo, Japan) compound microscope, as described by Samarakoon et al. [8].Where necessary, Melzer's reagent, Congo red, and Indian ink were used.The photographs included in the figures were edited using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA) and measured using the Tarosoft (R) Image Framework (v.0.9.7).
The herbarium specimens were deposited in the Chiang Mai University Herbarium (CMUB), Sustainable Development of Biological Resources Research Herbarium (SDBR), Center of Microbial Diversity, Sustainable Utilization, Department of Biology, Faculty of Science, Chiang Mai University, the Mae Fah Luang University Herbarium (MFLU), Chiang Rai, Thailand, and the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), Chinese Academy of Sciences, Kunming, China.New taxa were linked with the MycoBank (https://www.mycobank.org,accessed on 18 October 2023).

DNA Extraction, PCR Amplification, and Sequencing
DNA extractions were performed directly from the fruiting bodies.Total DNA extraction kits were used according to the manufacturer's instructions (PureDireX, Genomic DNA Isolation Kit, The BIO-HELIX Co., Ltd., New Taipei City, Taiwan).
Characters were assessed as unordered and equally weighted.The best evolutionary model for each gene was found using MrModeltest 2.3 and the Akaike Information Criterion (AIC).Phylogenetic trees were constructed using single and merged alignments of the genetic markers ITS, LSU, RPB2, and TUB2.Both maximum likelihood (ML) and Bayesian Inference (BI) methods were employed.The newly obtained sequences have been archived in GenBank for future research reference (Table 1).ML analyses were conducted using RAxMLGUI v.1.3[22], employing the ML+rapid bootstrap configuration with 1000 replicates.The Bayesian tree was constructed using MCMC sampling within MrBayes v3.1.2[23,24], comprising 1,000,000 MCMC generations utilizing four chains and partition analysis with 100 sampling frequencies.The initial 2500 trees (25% of the total) were designated as the burn-in phase and were subsequently excluded from the analysis.Posterior probabilities (PP) were calculated using the remaining 7500 trees.The generated trees were visualized using the FigTree v.1.4.0 program [25], and the final figures were crafted using Adobe Illustrator ® CS5 (Version 15.0.0,Adobe Systems, San Jose, CA, USA).

Phylogenetic Analyses
Based on the preliminary BLASTn search and morphological studies, new collections were identified into two distinct groups.Therefore, two phylogenetic analyses were conducted as core Xylariaceae and Xylariales genera incertae sedis for better resolution of the phylogenetic affinities.The overall tree topology for each analysis was consistent.All the gene regions resulted in the GTR + I + G model.The RAxML analyses of the combined ITS, LSU, RPB2, and TUB2 datasets yielded the best-scoring trees (Figures 1 and 2).Bayesian posterior probabilities from MCMC were evaluated when the final average standard deviation of split frequencies was less than 0.01.
Notes: Muscodor lamphunensis is similar to M. thailandica (MFLU 18-0784) and M. ziziphi (MFLU 18-0107) in having inconspicuous stromatic forms with rudimentary clypeus, 8-spored, unitunicate, cylindrical asci with a J+ apical ring, and uniseriate, naviculate to ellipsoidal, mostly hyaline, smooth-walled constricted apiosporous ascospores with remnant at both polars [12].Muscodor lamphunensis has larger ascomata and thick peridium compared to M. thailandica and M. ziziphi.On the substrate, we can observe a remarkably distributed black clypeus around the neck area in our collection.Muscodor lamphunensis and M. ziziphi share Type 1 paraphyses, as described by Samuels et al. [88].The ITS sequence of M. lamphunensis CMUB 40021 is similar to that of M. musae CMU MU3 (99%, 1/601 gaps) and M. albus MONT 620 (99%, 1/601 gaps), while the LSU sequence is similar to that of M. yunnanensis W-S-38 (97%, 6/1193 gaps), M. thailandica HKAS 102,323 (96%, 6/1200 gaps), and M. coffeanum MFLUCC 13-0159 (96%, 5/1125 gaps).Few RPB2 and TUB2 sequences are publicly available, and the species have not been identified among some of them.Therefore, the sequence comparisons are incomplete for the related taxa.The sequence comparisons of the RPB2 sequences from our new collection and the previously described three collections show significant differences as: M. thailandica MFLUCC 17-2669 (84%, 22/1086 gaps), M. ziziphi MFLUCC 17-2662 (83%, 22/1028 gaps), and M. coffeanum MFLUCC 13-0159 (85%, 16/1070 gaps).In our multigene phylogeny, two collections of M. lamphunensis (CMUB 40021 and MFLU 23-0408) cluster with M. musae JCM 18230, M. oryzae JCM 18231, and M. roseus MONT 2098 (Figure 1).However, the species segregation is not clear, probably due to the lack of other gene regions.There are no sexual or asexual morphologies of any species in this cluster.Since we failed to obtain a culture of this collection, the hyphal and culture morphologies are not possible.Even though M. musae JCM 18230 and M. oryzae JCM 18231 have been described from the same region, there is insufficient data to make any more comparisons.Based on the available morpho-molecular analyses, we introduce our new collection as a new species, M. lamphunensis from Thailand.However, it would not be surprising to find other species with asexual and sexual relationships, multigene phylogenies, and variable VOC profiles in the future, and to lump them together.[8].MycoBank: MB558737.Notes: Nigropunctata was introduced by Samarakoon et al. [8] to accommodate three new species, including the type species, N. bambusicola.Nigropunctata species are characterized by immersed ascomata; white or yellow ectostroma; cylindrical, short pedicel, apically rounded asci with J+, discoid or inverted hat-shaped apical ring, and cylindrical to broadly ellipsoidal, aseptate ascospores.Sugita et al. [89] introduced the Spirodecosporaceae to accommodate Spirodecospora, which has similar ascomata and asci morphologies to Nigropunctata.However, Spirodecospora species are characterized by broadly ellipsoidal to fusoid, aseptate, brown, verruculose ascospores with spirally or almost straight linear ornamentation.Interestingly, species from both Nigropunctata and Spirodecospora are described from bamboo substrates only.In this study, we introduced two new Nigropuntata species from Thailand, which are also collected from dead bamboo branches.

Discussion
New additions have occasionally altered the morpho-molecular taxonomy of the Xylariales/Amphisphaeriales/incertae sedis.Recent research on those incertae sedis taxa, which were previously known from a single collection, lacking recent collections, having inconsistent morphologies, no sexual-asexual links, and no molecular evidence, has urged the classification of genus to the family level.Families like the Appendicosporaceae [8], Barrmaeliaceae [32], Fasciatisporaceae [55], Gyrothricaceae [35], Oxydothidaceae [90], and Spirodecosporaceae [89] are some of the examples that have primarily been raised through studies on inconspicuous xylarialean taxa.Prior research has most likely focused on stromatic forms like diatrypoid, hypoxyloid, rosellinioid, and xylarioid rather than inconspicuous stromatic forms like anthostomelloid.The introduction of novel taxa does not adhere to these conservative approaches and emphasizes microscopic characteristics such as the type of ring, the color of the ascospores, and the presence or absence and type of germ slit [8].
Muscodor, Nigropunctata, and Xenoanthostomella are the three genera taxonomically identified during the study to focus on inconspicuous xylarialean taxa.If the stromatic nature was thought of as the key characteristic, those three genera would probably be treated as anthostomella-like taxa.Although incertae sedis taxa, particularly inconspicuous forms, are increasingly being split apart rather than being grouped because of molecular studies, we encountered several problems in each genus in their taxonomy and phylogeny.
With the addition of Muscodor brunneascosporus and M. lamphunensis, the number of species known from their sexual morphs has increased to five, which all are collected from northern Thailand [12,65].There is a clear differentiation of the ascospore characters in those species.However, obtaining the asexual morphs from cultures has failed; therefore, the sexual-asexual connection is unknown.Due to the fact that many of the available sequences are only ITS, the species delineation based on phylogeny is uncertain.Stadler et al. [91] discussed the intragenomic polymorphism of the ITS region of the Hypoxylaceae and revealed less than 97%, or around 90%, of the overall homology of the ITS sequences.The intragenomic ITS variation among Muscodor species or anthostomella-like taxa needed to be checked to understand the ITS based species demarcation.Nigropunctata and Spirodecospora species are associated with dead bamboo and are similar to inconspicuous stromatic forms [89].The microscopic morphologies and phylogenetic analyses support accepting them as distinct clades apart from the core Xylariaceae.This is important for the collection of fresh, inconspicuous xylarialean taxa and molecular phylogeny.
In summary, this study introduced five new xylarialean taxa (Muscodor brunneascosporus, M. lamphunensis, Nigropunctata hydei, N. saccata, and Xenoanthostomella parvispora), alongside the inclusion of M. coffeanus from northern Thailand.These species were all found on branches in varying stages of senescence and early decomposition during the rainy season.Nevertheless, the taxonomic classification of inconspicuous xylarialean taxa remains incomplete, and further investigations are needed, particularly with the inclusion of freshly collected specimens.The ongoing exploration of the taxonomy, phylogeny, and secondary metabolites of xylarialean taxa underscores northern Thailand's significance as a focal point for continued research.Consequently, our future studies will be directed towards comprehensive investigations encompassing morphology, phylogeny, ecology, and the exploration of antimicrobial properties for potential applications in plant disease control.

Figure 1 .
Figure 1.Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, RPB2, and TUB2 sequence data.The tree is rooted to taxa from the Barrmaeliaceae, Graphostromataceae, and Lopadostomataceae.Bootstrap support values for ML equal to or greater than 50%, PP equal to or greater than 0.9 (ML/PP) are given above or below the nodes.The newly generated sequences are in blue.Type collections are in bold.Analysis 2: The combined sequence alignment comprised 77 strains with 3612 characters, including gaps (ITS: 1-745, LSU: 746-1629, RPB2: 1630-2673, TUB2: 2674-3612).Single gene analyses were also performed, and topology and clade stability were compared from combined gene analyses.Tree topology from ML analysis was similar to that

Figure 1 .
Figure 1.Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, RPB2, and TUB2 sequence data.The tree is rooted to taxa from the Barrmaeliaceae, Graphostromataceae, and Lopadostomataceae.Bootstrap support values for ML equal to or greater than 50%, PP equal to or greater than 0.9 (ML/PP) are given above or below the nodes.The newly generated sequences are in blue.Type collections are in bold.

Figure 2 . 2 .
Figure 2. Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, RPB2, and TUB2 sequence data.The tree is rooted to the taxa belonging to the Sordariales.Bootstrap Figure 2. Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, RPB2, and TUB2 sequence data.The tree is rooted to the taxa belonging to the Sordariales.Bootstrap support values for ML equal to or greater than 50%, PP equal to or greater than 0.9 (ML/PP) are given above or below the nodes.The newly generated sequences are in blue.Type collections are in bold.Xenoanthostomella Mapook & K.D. Hyde, in Hyde et al., Fungal Divers.100: 235 (2020) [55].MycoBank: MB558737.Notes: Xenoanthostomella was introduced by Hyde et al.[55] with the type X. chromolaenae on Chromolaena odorata from Thailand and accepted in Xylariales, genera incertae

Table 1 .
Names, codes, and corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses of this study.